Dried blood factor composition comprising trehalose

ABSTRACT

A stable blood factor composition contains a stabilising amount of trehalose in the absence of human serum albumin to provide a product stable at up to  60  DEG C.

[0001] This invention relates to dried compositions of blood factors forreconstitution with water or aqueous solutions.

[0002] Blood factors, particularly factor VIII and factor IX, are nowthe standard treatment for diseases caused by a lack of the appropriatefactor, in particular haemophilia. The blood factor has generally beenderived from human blood by various extraction techniques, for exampleas disclosed in EP-A-0083483, or by expression in genetically modifiedmicro-organisms, for example as disclosed in EP-A-0160457 andEP-A-0182448.

[0003] Blood factor products such as factor VIII are highly delicate,unstable proteins. They are usually supplied in the form of frozensolutions in an appropriate buffer or, more generally, as freeze-driedpowders. Even the freeze-dried powders must be kept cold during storage.In order to stabilise the freeze-dried material, commercial productscontain a stabilising protein, in particular human serum albumin (HSA).It has not been thought possible to prepare a dry blood factorcomposition which is stable at ambient temperatures and atpasteurisation temperatures (e.g. 60° C.) in the absence of HSA.However, the presence of HSA introduces considerable problems ofpurification since it is essential that the protein is free of viralcontamination. The use of recombinant HSA to overcome these problems isexpensive.

[0004] Trehalose is known to be a highly effective stabilising agent fordelicate proteins, as disclosed in US-A-4891319, enabling proteins to bedried at temperatures above freezing. We have now found that iftrehalose is used to stabilise a blood factor product, not only can theproduct be dried with or without freezing, but also the product isstable even when retained at a temperature of 60° C. for an extendedperiod, in the complete absence of HSA. According to the presentinvention therefore we provide a stable dried blood factor compositioncontaining a stabilising amount of trehalose in the absence of albumin.

[0005] In general, any stabilising amount of trehalose may be used andan excess in general causes no problems. Indeed, the presence oftrehalose aids the rehydration process and is physiologically acceptablefor injection, being rapidly metabolised to glucose. In general a ratioof about 0.2 mg to 2.5 mg trehalose per unit of factor VIII isdesirable, especially 0.2 to 1.5 mg/unit. The composition isparticularly suited to formulations of factor VIII, which may alsocontain appropriate buffering and ion-reinforcing salts, in particular asource of calcium. In general, a ratio of about 1.0 to 1.5 μg of calciumions per unit of factor VIII is appropriate.

[0006] Other buffering and modifying agents may also be present in thedried material for reconstitution to the injection solution, for examplehistidine. However, we have found that the level of salts, particularlysodium chloride, present can affect the preservation on drying. It isthought desirable for the commercial product for injection to have anisotonic salt concentration. However, the processing formulations whichare freeze-dried are desirably hypertonic, typically containing about500 mM NaCl (isotonic NaCl =150 mM), as this is considered to helpstabilise the blood factor. As a result, commercial freeze-driedformulations generally have a high salt content and are reconstitutedfor injection with the appropriate amount of sterile water to obtain anisotonic solution.

[0007] A considerably reduced salt content is preferred for the driedmaterial of the invention and, in general a solution of about 500 unitsof Factor VIII per ml to be dried should preferably contain less than200 mM e.g. 75 to 150 mM, NaCl, especially about 100 mM., but can beeven lower, e.g. 20 to 50 mM, especially about 22 to 30 mM. Low saltpreparations possess a higher dry stability. The dried product can bereconstituted to the desired salt level with a saline solution insteadof the conventional water. In general, the molar ratio of trehalose tosalt should be above 1:1, especially above 2.5:1 e.g. above 10:1,preferably above 12.5:1.

[0008] The dried composition may be obtained by drying an appropriatesolution of the blood factor containing the correct proportions oftrehalose and other desired components. In general, the solution that isdried should simply contain all the components required in thereconstituted injection solution, although the solution for drying maynot necessarily be at the same dilution. Typically, the solution fordrying will contain from 1 to 1000 units of factor VRI per ml. Themethods of drying may include freeze drying, vacuum drying andspray-drying. A particularly preferred method according to the inventioncomprises vacuum drying at a temperature no greater than 25° C.,preferably no greater than 10° C., to form a foam, thus maximising theexposed surface and the drying effect. The following examples illustratethe invention further.

[0009] Example 1

[0010] Recombinant factor VIII was received as a deep frozen solutioncontaining approximately 2000 to 2500 units/ml in the manufacturer'shigh salt buffer. The thawed solution was dialysed against a buffersolution containing 500 mM NaCl, 15 mM CaCl₂ and 10 mM histidine at pH6.8. The dialysed protein was diluted in the same buffer, but with addedtrehalose, to give a final concentration of 500 units per ml and 10% byweight trehalose at pH 6.8. This solution was vacuum dried in 1 mlaliquots. Vacuum was reduced step-wise from atmospheric to 4 Pa (30mTorr) to avoid freezing the sample. The temperature of the sample wasnot allowed to rise above 12° C. until the formation of a foam, afterwhich the temperature was kept below 30° C. Total drying times were 24to 28 hours.

[0011] The samples were stored for 0, 1.5, 3 and 6 months at 40° C. andthen reconstituted in 5 ml aliquots of sterile distilled water beforebeing tested for activity. The results are shown in the following tablein comparison with a commercial freeze-dried product containing HSA.Both the test and commercial samples have a high salt content. Thepost-drying results show that with trehalose it is possible to dryfactor VIII successfully in the absence of HSA, but that a high saltcontent is unsatisfactory for long term storage, even in the presence ofHSA. Percentage of initial activity Time (months) Sample CommercialProduct 0 100.0 100.0 1.5 86.8 95.3 3 75.1 71.2 6 76.6 63.6

[0012] Example 2

[0013] Samples were dried as described in Example 1 but using a bufferformulation comprising 100 mM NaCi, 15 mM CaCl₂, 15 mM histidine and1.27 molar trehalose (43.5w/v) and stored at 60° C. beforereconstitution. The results are given in the following table, in whichthe activity is measured on an ACL 100 automated coagulometer(Instrumentation Laboratory SpA, Milan, Italy). The test sample, with alow salt content showed no significant loss of activity on storage, evenafter four weeks at 600° C. Percentage of initial activity recovered Wetcontrol 100.0 Post-drying 95.5 Two weeks 96.0 Four weeks 96.8

[0014] Example 3

[0015] Two formulations were prepared containing different saltconcentrations as shown: Formulation A Formulation B NaCl 0.13% NaCl1.03% CaCl₂ 0.011% CaCl₂ 0.011% L-histidine 0.12% L-histidine 0.12% Tris0.002% Tris 0.002% Tween 80 0.002% Tween 80 0.002% PEG 3350 0.004% PEG3350 0.004% Trehalose 7.5% Trehalose 7.5% Factor VIII 50 U/ml FactorVIII 50 U/ml Water to 100% Water to 100%

1. A stable dried blood factor composition containing a stabilisingamount of trehalose in the absence of albumin.
 2. A compositionaccording to claim 1 containing 0.15 to 2.5 mg trehalose per unit ofblood factor.
 3. A composition according to claim 1 or claim 2containing factor VIII.
 4. A composition according to claim 3 containing1.0 to 1.5 μg calcium ion per unit of factor VIII.
 5. A compositionaccording to any of claims 1 to 4 containing more than 2.5 molestrehalose per mole of sodium chloride.
 6. A composition according toclaim 5 containing more than 10 moles trehalose per mole of sodiumchloride.
 7. A method of preparing a composition according to any ofclaims 1 to 6, in which an aqueous solution of the blood factorcontaining trehalose is dried at a temperature no greater than 25° C. 8.A method according to claim 7, in which the solution is dried at atemperature no greater than 10IC.